objective The main objective of the present study is to isolate yeasts from different environmental sources to investigate their potential probiotic characteristics.Methods Appropriate in vitro assays has been conducted to examine their probiotic potentiality, such as acid and bile salt tolerance, temperature resistance, stimulated GIT tolerance, cell adhesion and cholesterol removal. All the yeast isolates were tested under in vitro conditions.Results In the present study, 20 yeast species have been isolated from different sources, screened and their desirable probiotic properties viz. pH tolerance, bile salt tolerance and thermo-tolerance have been evaluated. Screened yeast isolates treated with gastric juices showed increased survival rate above 60%. A further in vitro study investigates cholesterol removal and it showed increased cholesterol removal rate up to 92%. Exopolysaccharide production was estimated for selected yeast isolates and applications are under investigation.Conclusion Among 20 yeast isolates, 5 isolates showed increased growth under stress tolerance. It can be concluded that the screened yeast isolates LM, MR, GOI, GII2 and WI can serve as promising probiotics in various fields of food industry.Key words: Acid tolerance; bile tolerance, cholesterol removal, probiotics, yeast.